Immunodiagnosis of latent tuberculosis : new answers to an old question? Franken, W.P.J.

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(1)Immunodiagnosis of latent tuberculosis : new answers to an old question? Franken, W.P.J.. Citation Franken, W. P. J. (2009, June 10). Immunodiagnosis of latent tuberculosis : new answers to an old question?. Retrieved from https://hdl.handle.net/1887/13840 Version:. Corrected Publisher’s Version. License:. Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden. Downloaded from:. https://hdl.handle.net/1887/13840. Note: To cite this publication please use the final published version (if applicable)..

(2) 11 DOUBLE-BLIND RANDOMIZED PHASE I STUDY COMPARING RDESAT

(3) REAGENT IN THE DIAGNOSIS OF TUBERCULOSIS INFECTION. Sandra M. Arend1, Willeke P.J. Franken1, Henrik Aggerbeck2, Corine Prins1, Jaap T. van Dissel1, Birgit Thierry-Carstensen2, Pernille Nyholm Tingskov2, 2, Peter Andersen2. 1. Dept. of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands. 2. Statens Serum Institut (SSI), Copenhagen, Denmark. TUBERCULOSIS 2008;88:249-61.

(4) 178. Chapter 11. ABSTRACT

(5) !"!# $% &'!& * ! +$ $% in vitro assays based on Mycobacterium tuberculosis!"!&! that are lacking in Bacillus Calmette Guérin (BCG). In animal studies, intradermal /%&!!"!*$<$%&# was to assess the safety of intradermal recombinant dimer ESAT-6 (rdESAT-6) compared with tuberculin and to determine the human dose. The study design was a double-blind Phase I study with intrasubject randomization to the left and right forearm, comparing 2 Tuberculin Units (TU) intradermal tuberculin (RT23) with 0.01, 0.1, 1 or 10 μ $&$%"+#!$$$ tuberculosis (TB) patients. The risk of sensitization after skin testing was assessed in healthy volunteers. All doses were tolerated well by healthy volunteers and responses to rdESAT-6 were limited to transient redness after 24 h only at the highest dose. No sensitization was observed. Because 1 μg rdESAT-6 induced large responses with local side effects in some TB patients, the 10 μg dose of rdESAT-6 was not tested. Mean responses to 0.01, 0.1 and 1 μg rdESAT-6 measured 14.0, 19.8 and 38.8 mm of redness, respectively, and 7.0, 13.4 and 14.6 mm of induration. The response to tuberculin was similar to the responses to 0.1 μg rdESAT-6. Mild local side effects due to tuberculin and rdESAT-6 were observed in 8/15, respectively, 6/15 patients, more pronounced at the highest rdESAT-6 dose. In conclusion, this pilot Phase I study of safety, feasibility and $"$% $+$$%$%!$%!"!* test for human use. No serious adverse events were observed but the study was $ &%"!# $/ $ $ !$ %#< did not seem to sensitize healthy volunteers. In treated TB patients, responses to rdESAT-6 were optimal at 0.1 μg. Furtherstudies are needed to evaluate sensitization after repeated doses and to study the effectof additional CFP-10 on +#$%!"!*<.

(6) ESAT-6 versus tuberculin as skin test. 179. Tuberculosis (TB) remains the single most important fatal infection of humans, with 8.8 million new cases every year and an estimated 3 million deaths. Tuberculin $&"$++>??@K'&/$/%$$!&# as a skin test reagent to support the diagnosis of TB or for detection of latent TB infection (LTBI). The main disadvantage of the tuberculinskintest (TST) is the occurrence of false positive responses due to cross-reactivity to antigens in PPD that are shared by environmental mycobacterial species as well as by Bacillus 1,2 Calmette Guérin (BCG) vaccine strains. !"!# $% especially problematic in regions where BCG vaccination is standard practice and exposure to environmental mycobacteria is common, thus including most countries with high TB rates. !$+# $% !"! %$ Mycobacterium tuberculosis, being those lacking in BCG and most environmental mycobacteria, led to the development of in vitro assays based on antigen-stimulated interferon-γ (IFN-γ) 3,4 production. Various formats of such IFN-γ release assays (IGRA) showed a 5 +# # !$ !"!#< Two IGRA have thus far been ® marketed. QuantiFERON -TB Gold (QFT-G, Cellestis, Carnegie, Australia) is a whole-blood assay using ELISA for detection of IFN-γ responses. A novel intube version of QFT-G (QFT-GIT) contains an additional M. tuberculosis!"! TM peptide (TB7.7) and has logistical advantages. T-SPOT .TB (Oxford Immunotec, '$XK'$[#*&$$!\&>

(7) ?K and requires isolation of peripheral blood mononuclear cells. In various guidelines, ] +'!$+%$$!$"+ 6,7 following TST testing guidelines. During the past few years, IGRA are increasingly being used in various clinical– epidemiological settings for the detection of TB disease or LTBI, either in place of or as supplement to the TST. Compared to industrialized regions, the situation is different in less developed countries where the use of IGRA is limited by test costs and the need of laboratory facilities and trained personnel. In that setting, important issues are costs and practical feasibility even under critical circumstances such as interrupted power supply. * ' $ !"! /$& !$' + $% !"!#$%/$!$%*/$&$%'$$# facilities other than cold storage of the reagent. In vivo data of intradermal use of. Chapter 11. INTRODUCTION.

(8) 180. Chapter 11. !"! < $% '# " ! 8 infected with Mycobacterium bovis. Recombinant ESAT-6 elicited marked delayed type hypersensitivity skin reactions in guinea pigs infected with M. tuberculosis, but 9 not in those infected with BCG or Mycobacterium avium. Recently, a recombinant dimer of ESAT-6 synthesized in Lactococcus (recombinant dimer ESAT6 (rdESAT-6)) has been produced following good laboratory practice and good manufacturing practices to be tested in humans. In pre-clinical studies in guinea pigs, mice, rats and dogs, no adverse events were seen following subcutaneous or intravenous administration of one or several excessive doses of rdESAT-6 to 10 immunologically naïve animals. The aim of the present study was to evaluate the safety and diagnostic potential of intradermal rdESAT-6 in humans and to determine the human dose.. Subjects and methods Aims of the study Primary objective of the study was the assessment of the safety of four different doses of rdESAT-6 administered intradermally to healthy adult volunteers and to TB patients who had completed treatment. Secondary aims were to compare the safety and skin test responses to rdESAT-6 with 2 Tuberculin Units (TU) Tuberculin (RT23 SSI) as a reference. For healthy volunteers, the risk of sensitization to rdESAT-6 following a single intradermal administration was evaluated by comparing in vitro IFN-γ responses after ESAT-6 stimulation of whole-blood samples on days 2 and 28 after injection. In treated TB patients, in vivo responses to rdESAT-6 were compared with pre-study IFN-γ responses after in vitro ESAT-6 stimulation ® using the QuantiFERON -TB Gold assay.. Study design The study was designed as a single-center Phase I doseescalating clinical trial. The study was double-blind, within subject controlled and randomized between the right and left arm allowing simultaneous testing of rdESAT-6 and 2 TU PPD (RT23, SSI, Copenhagen, Denmark). The randomization was performed by a statistician at the Biostatistics Unit at SSI, who did not participate in the data management or the statistical analysis of data from the trial. Each subject received only one $$% < /"+$/$+><<^<^_.

(9) ESAT-6 versus tuberculin as skin test. 181. μ`^<_

(10) K$"+#&</$#$/$$!$_^ fold higher dose level if the previous dose level had been considered safe by the principal investigator (S.A.) and the data safety monitoring board. After testing of the highest dose level in the healthy volunteer group (i.e. 10 μ`^<_

(11) KX" treated TB patients were included at the lowest dose level (i.e. 0.01 μg/0.1 mL).. Study subjects. Chapter 11. &#${^#&$&$%"+&'\&# the same number of treated male or female TB patients. Before inclusion, pregnancy was excluded in women of childbearing age. General health was assessed by physical examination, routine blood and urine tests. Exclusion !/"!#'$'$$$&X!$% surface antigen, antibodies to HIV or hepatitis C virus, BCG vaccination within 5 years prior to the day of inclusion or BCG vaccination more than once, having !+/#X*$/&"!!#$%%! the lymphoid organs (e.g., Hodgkin’s disease, lymphoma, leukemia, sarcoidosis), use of immunosuppressive drugs within 3 months prior to the day of inclusion, vaccination with a live vaccine within 6 months prior to the day of inclusion or viral or bacterial infection at inclusion. To allow accurate evaluation of the size of the skin test reactions (induration and/or redness) subjects with black skin were not !&"+&$< Exclusion criteria for healthy controls were a history of TB, known contact to a ® person with active TB or a positive response to ESAT-6 in QuantiFERON TB Gold. TB patients were eligible if they had completed treatment and were without symptoms of active TB disease at the time of inclusion. Responsiveness of the treated TB patients to ESAT6 was assessed before inclusion using the ® QuantiFERON ]$#< "$&X$#$/$+ |γ response (according to the manufacturer’s instructions) after in vitro ESAT-6 stimulation were included. As bilateral skin test responses were observed already at the lowest dose level, the protocol was amended to allow for enrollment in &'\&$&$%%$!&&!$"'&/$ did not respond to ESAT-6 in vitro..

(12) 182. Chapter 11. Skin test reagents Both skin test reagents, rdESAT-6 and 2 TU Tuberculin PPD (RT23 SSI), were manufactured at SSI in Denmark according to Good Manufacturing Practice standards. All strengths of rdESAT-6 were dissolved in phosphate buffered saline containing 0.01% Tween and with pH adjusted to between 6.5 and 7.5. The rdESAT-6 vials in the four strengths and the PPD RT 23 vials could not be distinguished from each other visually and labels only indicated the side of injection as per randomization. The selected doses of 0.01–10 μg rdESAT-6 were based on results from pre10 clinical studies with rdESAT-6 in guinea pigs, mice, rats and dogs. A standard dose of 2 TU Tuberculin PPD RT23 SSI corresponds to approximately 0.04 μg protein. Estimates from pre-clinical studies in guinea pigs suggested that the injection of 10 TU PPD RT23 SSI and 0.024 μg rdESAT-6 result in skin responses of similar sizes. However, the relationship between responses to PPD and rdESAT-6 in humans was not known and the optimal dose of rdESAT-6 had to be established experimentally. Tuberculin and rdESAT-6 skin tests were administered intradermally following a standard protocol. All readings were independently performed by the principal investigator (S.A.) and research nurse (C.P.), who were trained and experienced in Mantoux testing. Discrepancies exceeding 2 mm were reassessed until consensus was reached. Measurements were done with calipers, and subsequently the distance between the calipers legs were measured with a ruler. On days 0 (skin test injection) through 4, digital images of both forearms including a size reference /*+!#%$"~!%$&$<.

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(14) Each trial subject was followed during 28 days after skin tests were administered (day 0, always on a Monday) with clinical observation during 2 h after injection and subsequently on days 1, 2, 3, 4 and 28. Both redness and induration were recorded because the characteristics of the response to the rdESAT-6 antigen in humans were not yet known, and redness could be a relevant parameter. Redness /"+'$*!$$$$%*$&!$ sites. The diameter of induration and/or redness at the injection sites was measured with calipers transversely to the long axis of the forearm at 24, 48, 72 and 96 h after injection and subsequently the distance between the calipers legs were.

(15) ESAT-6 versus tuberculin as skin test. 183. measured with a ruler. Local and systemic adverse reactions occurring within 28 days after application of the tests were recorded. Adverse events present on day 28 were followed up. Blood and urine parameters were retested on days 4 and 28. #!~$/$$#{<@"$$%++/ following the ICH E2A guideline (http://www.fda.gov/cder/guidance/ iche2a.pdf ). %!$&X%#$/$"@%# Monitoring Board of experts who advised on the safety of testing the next dose level. $&'$$##/%$'#!"!'$$# ® at LUMC according to standard procedures. The QuantiFERON -TB Gold 2nd generation assay using soluble ESAT-6 was performed at the laboratory of the Department of Infectious Diseases according to the manufacturer’s instructions.. Statistical analysis Descriptive statistical methods were used. The study was designed as an initial pilot study on safety and proof of principle of intradermal rdESAT-6 and the numbers /$$$!"!!</##$% variance (ANOVA) was used to evaluate the effect of different parameters on the difference between the independent skin test measurements. Correlation between in vivo and in vitro responses was analyzed by Spearman correlation.. Role of the funding source The study sponsor was involved in the study design and participated in the writing of the report. The study sponsor had no role in the collection, analysis, and interpretation of data or the decision to submit the paper for publication.. Results. /##&%!+$&_$&$%"+ subjects were included on different dates, each new group being included after the previous dose level had been considered safe. Characteristics of participants are shown in Table 1. Between 0 and 2 subjects in each group had been BCG vaccinated, which was similar for patients and controls. The average age of treated TB patients was higher than the age of the controls, which was related to the recruitment procedure for healthy uninfected controls that mainly attracted. Chapter 11. Study participants.

(16) 184. Chapter 11. students. TB patients were recruited without selection criteria other than past TB diagnosis and completed treatment. They had been diagnosed with pulmonary (N = 5), pleural (N = 3), other extrapulmonary (N = 5; arthritis, spinal TB, peritonitis, brain abscess, renal) or disseminated TB (N = 2) between 3 and 60 years before the start of the present study. Nine of 15 cases were microbiologically proven. Four patients had a clinical diagnosis of TB (pulmonary TB in a technician after laboratory exposure, skin test conversion in association with pleuritis in a contact of a smear-positive colleague, Moroccan man with classical thoracic spine lesions, &$# " $% $& / $+ K %$& responded favorably to TB treatment only. Two patients had not been treated with tuberculostatic drugs, because those were not available at the time of diagnosis, and reported spontaneous recovery of the disease. No clinical documentation was available for these two patients but the clinical presentation had been compelling, '$ / $+ &| ]$ "! * responses to rdESAT-6. All patients were without clinical signs or symptoms of TB disease at the time of the study.. Skin test responses Skin test responses to tuberculin and rdESAT-6 at four dose levels are shown in Figures 1 and 2 and Table 1. In healthy controls no responses were observed to 0.01 μg rdESAT-6. After injection of 0.1 or 1 μ X $ $% "+ &'! per group had transient local redness of 3 and 1 mm, respectively. The highest dose of 10 μg rdESAT-6 induced a transient redness of 19.4±7.1 mm after 24 h in 5/5 healthy volunteers that was not associated with local complaints and had dissolved completely at 48 h (Figure 3A). Skin test responses to tuberculin were observed in several controls, including three BCG vaccinated (7, 9 and 10 mm induration) and one unvaccinated subject (9 mm induration). In treated TB patients, the average of the maximal redness in response to 0.01, 0.1 and 1 μg rdESAT-6 was 14.0±10.4, 19.8±12.6 and 38.8±29.1, respectively. The corresponding indurations were 7.0±6.6, 13.4±8.5 and 14.6±11.2 mm, respectively. The differences between skin test responses in the three dose groups did not !+$%"!!X'&$+/$ and considerable local complaints (see below under Safety). Strong responses to rdESAT-6 were observed in both patients who had never received antibiotic treatment of TB (redness/induration of 29/21, respectively, 63/20 mm, compared to 29/14, respectively, 22/11 mm in response to 2 TU tuberculin). Notably, one patient in each of the three groups of treated TB patients was nonresponsive to.

(17) ESAT-6 versus tuberculin as skin test. 185. rdESAT-6 two of whom had culture-proven diagnosis and were Quantiferon TB Gold positive. Responses to 2 TU tuberculin were similar in the three groups of TB patients that received different doses of rdESAT-6. Overall, skin test responses to 0.1 μg rdESAT-6 were almost equivalent to those to 2 TU of tuberculin (Figures 2 and 3B and Table 1), redness and induration being alike. At the time of the study, the investigators were blinded as to the allocation of rdESAT-6 and tuberculin to the left or right arm. During the study, it was noted that the characteristics of &$$'$/%%/!$"'$ of induration, with one side feeling softer and the border being more gradually $!$$"&$/"'$$!$ side. These characteristics were not part of the study design, however, and were therefore not consistently recorded.. Kinetics of skin test responses Maximal redness in response to rdESAT-6 in TB patients was generally observed after 48 or 72 h, which roughly coincided with maximal redness in response to tuberculin. Indurations in response to rdESAT-6 peaked after 48–96 h in different patients. In patients with a maximal induration at 96 h, somewhat smaller but still comparable responses were also observed at 48–72 h. Most patients with maximal responses after 48 or 72 h had slightly smaller responses at 96 h, except for one patient with a 10 mm induration after 48 h which had decreased to 1 mm at 96 h. The optimal read-out time for both skin tests thus appeared to be 48–72 h after injection.. The two independent readings of skin test responses were highly correlated; !$$ !$%"! %$ /$ & / 0.972 for redness and 0.965 for induration (P<0.001). After exclusion of all readings that were zero for both readers, the correlation was 0.938 for redness and 0.934 for induration (P<0.001). There was no digit preference of readings. The size of the difference between the independent readings of redness as well as of induration were only associated with being a control or TB patient (P<0.001 both for redness and induration), but not with the injected substance being tuberculin or rdESAT-6, the side of injection, the rdESAT-6 dose level, the skin type (white or light brown), or the day of reading. After exclusion of all readings that were zero %$'$X&'!!$#/$$"!<. Chapter 11. Inter-reader variability of skin test responses.

(18) 5 5 5 5 20. 5 5 5 0 15. HC 1 HC 2 HC 3 HC 4 All HC. TB TB TB TB All. 0.01 0.1 1 10. 0.01 0.1 1 10. Dose rdESAT-6 (mg). 3 (20). (20) (20) (40) (20). 57.6 (32–78). 0 1 1 2 4 1 (20) 1 (20) 1 (20). (19–58) (19–45) (20–26) (20–25) (19–58). No. (%) BCG vaccinated. 63.8 (41–75) 46.2 (32–76) 62.9 (45–78). 30.1 27.7 23.1 22.9 25.9. Avg (range) age (yr). Median 12 (3–60). 3, 4, 5, 16, 20 5, 8, 12, 15, 43z 4, 8, 13, 30, 60z. Years since diagnosis TB. (0–3) (0–1) (8–24) (0–24). 24.2 (0–63). 14.0 (0–27) 19.8 (0–30) 38.8 (0–63). 0 0.6 0.2 19.4 5.1. 11.7 (0–30). 7.0 (0–14) 13.4 (0–21) 14.6 (0–30). 0 0.2 (0–1) 0 0 0.05 (0–1). (0–2) (0–16) (0–15) (0–16). 23.4 (0–8). 27.8 (11–43) 23.6 (0–48) 18.8 (12–24). 0 0.4 3.2 9.4 3.3. 13.0 (0–21). 13.4 (1–21) 13.0 (0–21) 12.6 (8–18). 0 0 1.8 (0–9) 5.6 (0–10) 1.9 (0–10). Induration. Redness. Redness. Induration. Avg (range) of maximal individual responses to 2 TU PPD (mm)y. Avg (range) of maximal individual responses to rdESAT-6 (mm)y. Abbreviations: BCG: M. bovis bacille Calmette Gue ´rin, HC: healthy uninfected controls, TB: treated TB patients. Kruskall–Wallis test for comparison of the time since diagnosis TB between the three groups of TB patients not signiﬁcant (P ¼ 0.53). y Data are expressed as the average (range) of the maximal individual responses observed during the 5 days after injection. z Denotes the interval since TB was diagnosed in the pre-antibiotic era in two patients. y This group was not tested.. 1 2 3 4y TB. No.. Characteristics of study population and skin test responses to tuberculin and rdESAT-6.. Group. Table 1. 186 Chapter 11.

(19) ESAT-6 versus tuberculin as skin test 0.01 mcg rdESAT-6. 30 Induration (mm). 20 10. 0. 48. 72. 96. 0. 24. 48. 72. 96. 0. 24. 48. 72. 96. 0. 24. 48. 72. 96. 0. 24. 48. 72. 96. 30 Induration (mm). Redness (mm). 24. 0.1 mcg rdESAT-6. 30 20 10 0. 20 10 0. 0 30. 24. 48. 72. 96 30. 1 mcg rdESAT-6. Induration (mm). Redness (mm). 10 0. 0. 20 10 0. 20 10 0. 0 30. 24. 48. 72. 96 30. 10 mcg rdESAT-6 Induration (mm). Redness (mm). 20. 20 10 0. 20 10 0. 0. 24. 48. 72. h after injection. 96. h after injection. Figure 1 Skin test responses to rdESAT-6 and tuberculin in healthy controls. Average (SD) skin test responses as redness and induration during 4 days after injection of 2 &'!&>$K >$K$&$%"+#&%! controls per dose level of rdESAT-6.. Chapter 11. Redness (mm). 30. 187.

(20) 188. Chapter 11. 0.01 mcg rdESAT-6. 30 Induration (mm). Redness (mm). 30 20 10 0. 24. 48. 72. 96. 0.1 mcg rdE SAT-6. 0. 24. 48. 72. 96. 0. 24. 48. 72. 96. 0. 24. 48. 72. 96. 30 Induration (mm). 30 Redness (mm). 10 0. 0. 20 10 0. 20 10 0. 0. 24. 48. 72. 96. 1 mcg rdESAT-6. 60. 30. 50. Induration (mm). Redness (mm). 20. 40 30 20 10. 20 10 0. 0 0. 24. 48. 72. h after injection. 96. h after injection. Figure 2 Skin test responses to rdESAT-6 and tuberculin in treated TB patients. Average (SD) skin test responses as redness and induration during 4 days after !$ $% { &'!& >$ K >$ K $& $% "+ treated TB patients per dose level of rdESAT-6. Note that the Y-axis for redness in treated TB patients injected with 1 μg rdESAT-6 is expanded to 60 mm..

(21) 189. Chapter 11. ESAT-6 versus tuberculin as skin test.

(22) 190. Chapter 11.

(23) ESAT-6 versus tuberculin as skin test. 191. Figure 3 Examples of skin test responses. (A) Representative example of redness in response to 10 μg rdESAT-6 at 24 h that has disappeared at 48 h in a BCG unvaccinated healthy control (right forearm). In comparison, a response to 2 TU RT23 is seen at 48 h (left forearm) that was maximal at 72 h (not shown). (B) Comparable responses to 0.1 μg rdESAT-6 and 2 TU PPD RT23 in a TB patient. (C) Hyperresponsiveness to 1 μg rdESAT-6 in a patient who had suffered from TB 60 years earlier. The maximal area of redness at 72 h measured 63 mm transversely and 123 mm longitudinally, induration was 20 mm (the inlay shows wristwatch impression resulting from edema. Chapter 11. of the forearm)..

(24) 192. Chapter 11. Safety Local side effects are listed in Table 2. Healthy uninfected controls all tolerated intradermal tuberculin as well as each of the four dose levels of rdESAT-6 without local complaints or changed laboratory parameters, except for the occurrence of mild local pain 24 h after injection of 10 mg rdESAT-6 in one subject that did not require the use of analgetics. In TB patients, bilateral skin test responses were already observed at the lowest dose level of rdESAT-6 and mild itch in one person was the only adverse event. At the second dose level responses were comparable in size on both arms, with similar side effects of itch or mild pain. After injection of 1 μg rdESAT-6, local responses were clearly larger and caused considerable local complaints including itch, pain and edema of the forearm in two patients and axillary lymphadenitis and vesiculation in one patient each (responses of one patient are shown in Figure 3C). Based on these observations, it was decided not to proceed to the highest dose level of 10 μg rdESAT-6. After deblinding, the largest responses causing $%%!/!$"$'$_μg dose of rdESAT-6..

(25) 5 5 5 5. 1 2 3 4. 1 2 3 4z. HC HC HC HC. TB TB TB TB. 0.01 0.1 1 10. 0.01 0.1 1 10. Dose rdESAT-6 (mg). 1/1 3/5 2/6. 0 0 0 0 2/3 3/7 3/7. 0 0 0 0 0 2/5 2/4. 0 0 0 1/1. rdESAT-6. rdESAT-6. PPD. Pain. Itch. 1/1 3/6 2/3. 0 0 0 0. PPD. 0 0 1/2. 0 0 0 0. rdESAT-6. 1/4 0 0. 0 0 0 0. PPD. Vesiculation. 0 0 1/3. 0 0 0 0. rdESAT-6. 0 0 0. 0 0 1/1 0. PPD. Axillary lymphadenitis. 0 0 2/4y. 0 0 0 0. rdESAT-6. Other. 0 1/1z 0. 0 0 1/1y 0. PPD. Chapter 11. Abbreviations: HC; healthy controls; PPD; puriﬁed protein derivative; rdESAT-6: recombinant dimer ESAT-6; TB: tuberculosis patients. The occurrence of side effects is expressed as number of individuals per group experiencing the side effect during the study/sum of the number of days that side effect was experienced per group (e.g. two subjects experienced side effect during 3, resp. 1 day is expressed as 2/4). y Short-lasting stiffness of the muscle proximal to the injection site. z Tired feeling of the whole R arm, without loss of function. y One patient experienced a tingling sensation at the injection site, edema of the forearm in association with itch, pain necessitating use of acetaminophen during 1 day and vesiculation lasting 5 days. The other subject experienced extensive edema of the forearm during 2 days (see inset in Figure 3C), in association with itch, mild pain that did not require treatment and axillary lymphadenitis. z This group was not tested.. 5 5 5 0. No.. Group. Table 2 Side effects caused by intradermal rdESAT-6 or tuberculin (scored as number of persons per group suffering from the side effect/sum of duration in days of the adverse event of all subjects per group).. ESAT-6 versus tuberculin as skin test 193.

(26) 194. Chapter 11. In vitro responses to ESAT-6 To assess the risk of sensitization by intradermal administration of rdESAT-6 to healthy uninfected controls, in vitro whole-blood responses to ESAT-6 were measured by Quanti-FERON TB Gold at 48 h and 4 weeks after injection of the skin tests. Test results of all 20 controls were negative at both follow-up time points indicating that one injection of rdESAT-6 up to 10 μg did not induce a positive in vitro T cell response to ESAT-6. Of note, only one injection of rdESAT-6 was given per individual thus in vivo sensitization induced by repeated testing was not evaluated. "$&$%$+&| ]$$X as indicated in the protocol. Coincidentally responses in this group were higher (10.9±5.5 IU/mL IFN-γ), than responses in the subsequent groups (1.2±1.0, resp. 1.72±1.86 IU/mL). Two patients in the two higher dose groups had negative preskin test QuantiFERON TB Gold responses to ESAT-6 (0.19 resp. 0.28 IU/mL IFN-γ, cut-off≥0.35 IU/mL). The responses could not be determined in one patient in each of the dose groups 0.1 and 1 μg rdESAT-6 due to a failing NIL response and a high background, respectively. In TB patients, there was no statistically "!$!$'/*in vitro response to ESAT-6 and skin test responses to rdESAT6 as redness or induration at any dose level or for combined groups (Figure 4). Three of four patients with a negative or indeterminate in vitro response at baseline had large-sized skin test responses (≥10 mm induration) to rdESAT-6. Of three patients with no skin test response to rdESAT-6 whatsoever, two had culture-proven diagnosis and had a positive QuantiFERON TB Gold response to ESAT-6 (11.83 and 0.52 IU/mL, respectively) and one was Quanti-FERON TB Gold negative but close to the cut-off level (0.28 IU/mL) in the dose groups of 0.01, 0.1 and 1 μg rdESAT6, respectively. The latter patient was $&$%$/$+&$#" in association with TST conversion responding to TB treatment. QuantiFERON TB Gold using ESAT-6 as well as QuantiFER-ON TB Gold intube measuring responses to the combination of ESAT-6, CFP-10 and TB7.7 /#{%*"+/+$ indeterminate pre-skin test in vitro responses. Three now had a strong positive response, similar for both QuantiFERON formats (data not shown)..

(27) Redness in response to rdESAT-6 (mm). ESAT-6 versus tuberculin as skin test. 70 60 50 40 30 20 10 0 0. Induration in response to rdESAT-6 (mm). 195. 1 2 3 4 5 5 20 In vitro IFN-gamma response to ESAT-6 (IU/mL). 25. 20. 15. 10. 5. 0 0 1 2 3 4 5 5 20 In vitro IFN-gamma response to ESAT-6 (IU/mL). Figure 4 Relation between in vitro responses to ESAT-6 and in vivo skin test responses to rdESAT-6. Each data point depicts the in vitro response to ESAT-6 of one TB patient on the X-axis with the corresponding redness (A) or induration (B) in dose level. Symbols represent redness (open symbols) or induration (closed symbols) in the different rdESAT-6 dose groups as follows: ({) redness (0.01 μg rdESAT-6); (z) induration (0.01 μg); (՛) redness (0.1 μg); (W) induration (0.1 μg); () redness (1 μg); () induration (1 μg).. Chapter 11. response to intradermal rdESAT-6 on the Y-axis. Five patients were tested at each.

(28) 196. Chapter 11. Discussion

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(30) that intradermal injection of rdESAT-6 was safe, that a single dose up to 10 μg did not sensitize uninfected subjects as measured by in vitro T cell responsiveness to ESAT-6 and that injection of 0.1 μg elicited a response in treated TB patients that was similar to 2 TU of tuberculin. No skin test responses to rdESAT-6 up to 1 μg were observed in healthy controls including those who were BCG vaccinated.

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(32) human use. For developing countries a skin test has obvious advantages over in vitro tests such as IGRA and most importantly no need for laboratory facilities. A major limitation of the TST is the occurrence of false-positive responses following BCG vaccination or infection with environmental mycobacteria and that limitation would be overcome with a skin test based on ESAT-6 and/or other M. tuberculosis worldwide most individuals still receive BCG vaccination after birth or at school age. The only expected source of cross-reactivity of human relevance could be the rare infections with Mycobacterium kansasii or Mycobacterium marinum that 10,11 can induce responses to ESAT-6. Based on the results of this study, it was concluded that 0.1 μg ESAT-6 was well tolerated, induced responses at 48–72 h of a size similar as those to tuberculin and therefore seems the optimal human dose for intradermal use, in agreement with 10 the predicted human dose based on data obtained in animal studies. Our study

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(44) of redness after 24 h at the 10 μg dose level that had disappeared completely after 48 h. This high dose was not tested in TB patients because the 1 μg dose already induced very large responses and local complaints. Thus, it is conceivable that any skin test response to 0.1 μg rdESAT-6, be it induration or redness, observed at 48–72 h can be regarded as relevant. However, whether induration or redness are eventually chosen as a read-out is still open for debate. The measurement of induration is notoriously unreliable and requires training and experience, whereas.

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(49) Three TB patients in our small study failed to respond to intradermal rdESAT-6 although being in vitro responsive to the antigen and vice versa. The biological background for this difference is unclear and our study was not designed to address such discrepant responses but it may relate to the different homing

(50) !"# 6. In this regard, discrepant positive TST with negative IGRA results have been associated with higher age, indicating that IGRA may be less sensitive to detect 12 infection that was not recently acquired. It is possible that the relatively short incubation time of 24 h that is used in IGRA allows detection of effector T cell.

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(55) incubation times indeed increased sensitivity to more than 90% in subjects that 13,14 In this context, a skin test may have were negative in a short incubation IGRA. the advantage that the antigen is available for recognition and immune stimulation for three as opposed to one day. This may explain the observation that, in contrast to IGRA, the TST has documented high sensitivity for recent as well as remote 1,2 infection with M. tuberculosis. This is in agreement with the data from our study demonstrating that rdESAT-6 induced skin test responses in patients who suffered from TB between 3 and as long as 60 years earlier. There is an ongoing discussion $&'#

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(58) 15 latent infection. Although more data on the performance of ESAT-6 based IGRA versus skin test in different clinical groups are needed, our data suggest that a novel M. tuberculosis

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(60) of LTBI. The idea of this study originated with the successful introduction of IGRA for immunodiagnosis of infection with M. tuberculosis. Measured by the number of recent publications, IGRA provide a much-wanted alternative to TST. However, $&'#

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(62) 15 at least some of those. In high-endemic and low-resource settings, the use of $&'#

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(64) of the assay, lack of laboratory facilities and skilled personnel. The need of a blood sample has been named as a disadvantage especially in younger children and high rates of indeterminate results have been found in children and immunosuppressed 16,17 Most importantly, the detection and treatment of LTBI has thus far not adults. been part of TB control policies in most high-endemic regions where the TST is almost uniformly positive and does not discriminate between BCG vaccination

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(67) . 197. Chapter 11. ESAT-6 versus tuberculin as skin test.

(68) 198. Chapter 11. paper treatment of the immense numbers of latently infected individuals in these regions would be virtually impossible with existing resources and on a global level preventative therapy is recommended by World Health Organization only for 18 HIV-infected persons with LTBI or other individuals at high risk of TB disease. A prognostic marker for TB disease that would allow targeted treatment of the small proportion of latently infected individuals in high-endemic regions who are at highest risk for developing TB disease is therefore an international research 19 priority. A report by Doherty et al. has thus far remained the only indication based on clinical data that a positive IGRA result predicts progression to TB disease during a 2-year follow-up period, but data from a range of animal models have provided strong evidence for the correlation between high ESAT-6 responses and 20–22 The results of several large ongoing follow-up studies, subsequent disease. as summarized in Ref. 23 may provide more solid evidence for ESAT-6 based prediction of disease and pave the road for a skin test based on M. tuberculosis23

(69) It remains to be determined whether recombinant proteins are optimal for skin testing. In an in vitro culture system, overlapping peptides induced identical 24 responses as recombinant ESAT-6. In guinea pigs, individual peptides were compared with recombinant ESAT-6 for skin testing and skin test responses to the 9 N-terminal peptide were observed. However, the use of overlapping peptides for a human skin test would have disadvantages compared with recombinant proteins with regard to the costs of large-scale production and complexity of quality control. In addition, it is not known whether small-sized peptides might diffuse from the injection site precluding local skin test responses. Our study used rdESAT-6 as a

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(71) )*+:; most likely be necessary for maximal sensitivity, as has been demonstrated for IGRA. In guinea pigs, synergy was observed when ESAT-6 was combined with 9 MTP-64 in a skin test. An important issue concerns the development of novel TB vaccines. Currently, ESAT-6 is tested as a component of a hybrid vaccine. The use of an antigen as vaccine component would preclude its use for diagnostic purposes. However, it is expected that ESAT-6 can be substituted for other immunogenic antigens,

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(74) for diagnostic purposes. A limitation of our study was the small number of study subjects and the inclusion of only adult TB patients all but two with completed treatment. The power of the.

(75) ESAT-6 versus tuberculin as skin test. 199. 25. study was therefore too low to exclude uncommon side effects. Further studies should evaluate the safety and test performance in patients with untreated TB disease and those with recently acquired or past LTBI, including pregnant women and children. Repeated intradermal administration of a protein antigen can induce

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(78) use, e.g. in population surveys or yearly screening of risk groups, and should not sensitize uninfected subjects even after repeated use as was observed with high 10 doses and short dosing intervals in a guinea pig model. The risk of sensitization of uninfected humans is currently being studied by repeated administration of rdESAT-6. $ + $

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(85) use. No serious adverse events were observed among 20 uninfected subjects

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(89) complete safety. In TB patients, a dose of 0.1 μg induced a skin test response >?@

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(91) studies of safety, the risk of sensitization after repeated use and the value of additional antigens.. The authors thank all study subjects for their participation. Paul F. Douw van der J K K

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(94) & Department of Radiology at Leiden University Medical Center provided invaluable technical information and suggestions regarding digital imaging of the skin test responses. Funding: The study was sponsored by Statens Serum Institut, Copenhagen, Denmark (Trial code: TESAT-01, EUDRACT No.: 2005-001850-26). The sponsor participated in the design of the study. The sponsor monitored the conduct of the study, data collection and data management. The sponsor had no role in the analysis and interpretation of the data, the preparation, review, or approval of the manuscript. Ethical approval: The study protocol (EUDRACT No.: 2005001850-26, LUMC protocol No: 05.083), the Investigator’s Brochure and the Investigational Medicinal Product Dossier and all remaining documents were approved by the Ethical Review Board of LUMC. All subjects gave informed consent for blood sampling and skin testing after written information was provided.. Chapter 11. Acknowledgments.

(95) 200. Chapter 11. Data Safety Monitoring Board: The Data Safety Monitoring Board consisted of Dr. H.B. Thio, MD, PhD, (Head) Department of Dermatology, Erasmus Medical Center, Rotterdam, The Netherlands (h.thio@erasmusmc.nl); Dr. H.C. Rumke, PhD, Vaccine Center Rotterdam, Rotterdam, The Netherlands (h.rumke@erasmusmc. \

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(97) University Medical Center Groningen, The Netherlands, (c.g.m.kallenberg@int. umcg.nl). Competing Interests: "# ^* )+

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(100) ""$!"# 6 and sponsored the study. Contributions:"#J^

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(108) ~ S.A. and C.P. recruited and screened the study subjects, S.A., W.F. and C.P. conducted the study; S.A. photographed all skin test responses; W.F. was responsible for the blood tests; S.A. documented all clinical data and study results; B.T. and P.N. were study monitors; S.A., W.F. and H.A. analyzed the data; S.A. ~ ^* |# )+ _K +} J^

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(112) S.A. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.. Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tube.2007.11.004.. References (1). Huebner RE, Schein MF, Bass Jr. JB. The tuberculin skin test. Clin Infect Dis 1993;17:968–75.. . ?\. ]>'$ $`'(|

(113) !"

(114) Tuberculosis, a comprehensive international approach. 2nd ed. New York: Marcel Dekker; 2000. p. 279–322.. . \. #

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(116) diagnosis of tuberculosis. Lancet 2000;356:1099–104.. (4). Pai M, Riley LW, Colford Jr. JM. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004; 4:761–76..

(117) ESAT-6 versus tuberculin as skin test. . =\. 201. + ] J " K

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(119) diagnosis of tuberculosis: part I. Latent tuberculosis. Expert Rev Mol Diagn 2006;6:413–22.. (6). Mazurek GH, Jereb J, Lobue P, Iademarco MF, Metchock B, et al. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR Recomm Rep 2005;54:49–55.. (7). National Collaborating Centre for Chronic Conditions. Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control. London: Royal College of Physicians. Available at: /http://www. nice.org.uk/page. aspx?o=CG033 S. Accessed August 27, 2007.. . \. + _] ]} _ | )

(120) _+ ) ! " delayed-type hypersensitivity responses to ESAT-6 identify tuberculosisinfected cattle. J Clin Microbiol 2003;41: 1856–60.. (9). Elhay MJ, Oettinger T, Andersen P. Delayed-type hypersensitivity responses to ESAT-6 and MPT64 from Mycobacterium tuberculosis in the guinea pig. Infect Immun 1999;66:3454–6.. (10) AggerbeckH, Madsen SM. Safety of ESAT-6. Tuberculosis (Edinburg) 2005;86:363–73. . ::\ #

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(124) + !)"K et al. Tuberculin skin testing and in vitro T cell responses to ESAT-6 and. . . :; ] ] kansasii. J Infect Dis 2002;186:1797–807.. (12) Arend SM, Thijsen SF,Leyten EM, Bouwman JJ, Franken WP, et al. Comparison of two interferon-g assays and tuberculin skin test for tracing tuberculosis contacts. Am J Respir Crit Care Med 2007;175:618–27. (13) Cehovin A, Cliff JM, Hill PC, Brookes RH, Dockrell HM. Extended culture enhances sensitivity of a gamma interferon assay for latent Mycobacterium tuberculosis infection. Clin Vaccine Immunol 2007;14:796–8. (14) Leyten EM, Arend SM, Prins C, Cobelens FG, Ottenhoff TH, et al. Discrepancy . . ] γ release assays using short versus prolonged in vitro incubation. Clin Vaccine Immunol. :=\ +]K

(125) J) _"* ' diagnosis of latent tuberculosis infection: moving the research agenda forward. Lancet Infect Dis 2007;7:428–38.. Chapter 11. 2007;14:880–5. .

(126) 202. Chapter 11. (16) Ferrara G, Losi M, D’Amico R, Roversi P, Meacci M, et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet 2006;367:1328–34. (17) Connell TG, Curtis N, Ranganathan SC, Buttery JP. Performance of a whole blood interferon gamma assay for detecting latent infection with Mycobacterium tuberculosis in children. Thorax 2006;61:616–20. (18) Preventive therapy against tuberculosis in people living with HIV. Wkly Epidemiol Rec 1999;74:385–98. (19) Doherty TM, Demissie A, Olobo J, Wolday D, Britton S, et al. Immune responses . . ] !"#< infection among contacts of tuberculosis patients. J Clin Microbiol 2002;40:704–6.. (20) Dietrich J, Aagaard C, Leah R, Olsen AW,. Stryhn A, et al. Exchanging. ESAT6 with TB10.4 in an Ag85B fusion moleculebased tuberculosis subunit . . `

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(131) *!+_]K between Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle by using recombinant mycobacterial antigens. Clin Diagn Lab Immunol 1999;6:1–5. (22) Vordermeier HM, Chambers MA, Cockle PJ, Whelan AO, Simmons J, et. . . ). !"#<

(132) in cattle following Mycobacterium bovis BCG vaccination against experimental bovine tuberculosis. Infect Immun 2002;70:3026–32.. . ?\ #

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(136) J!K_]. . . . . tuberculosis: can disease be predicted? Trends Mol Med 2007;13:175–82. Antigenic equivalence of human T-cell responses to Mycobacterium 'K:

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